Symposium on Clinical Laboratory Medicine

Laboratory Diagnosis of Pleural and Peritoneal Effusions K. W. Prasse, D.V.M., Ph.D.,* and J. R. Duncan, D.V.M., Ph.D.t

The occurrence of an effusion in one or both body cavities is a frequent finding in certain diseases of the dog and cat. Using rather simple and routine laboratory procedures these effusions may be categorized by the clinician as pure transudate, modified transudate, nonseptic exudate, septic exudate, or hemorrhage, and in a few instances a more definitive diagnosis may be rendered. This classification of effusions is broader and more clinically useful than the traditional characterization as transudate or exudate. Certain transudates have visual or physiochemical findings traditionally typical of exudates yet their formation or pathogenesis is that of a transudate; these are modified transudates (of which several types occur) and can be identified by the routine employment of a complete evaluation procedure. The collection of body cavity effusions 4 • 7 •8 •9 and the examination procedure 6 have been described in a previous issue of the Veterinary Clinics of North America. The routine examination procedure includes visual inspection, total nucleated cell count (as per white blood cell counting methods), refractometry of the centrifuged supernatant fluid (specific gravity and estimated protein concentration), and microscopy of stained smears prepared directly from fresh fluid and from sediments following centrifugation. Other procedures such as urea nitrogen, bilirubin and globulin chemistries, or bacteriologic cultures may be employed as needed. This paper reviews the cytology of effusions and presents methods of differential diagnosis. *Associate Professor, Department of Veterinary Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia 7Professor, Department of Veterinary Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia

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Figure I . Transudates or m odified transudates (Wright's stai n , x 1200.) A , Reac t ive mesothelial cell (top ) with eosinophilic brush borde r a nd red blood cells. B, Binucleated r eactive mesothelial cell , small lymphocyte , and red blood cells. C, Vacuolated transfor m ed mesothelial cells and red blood cells . D, Reactive an d transformed mesothelial cells with so me intermediate forms and red blood cells. E , An aggr egate of r eactive mesothelial cells, tr ansformed m esothelial cell (bottom) , no ndegener ate neu tro phils, a nd r ed blood cells. F , Mesothelial cell sheet collected by ple u ral imprint. G, One mitotic and one trinucleated reactive mesothelial cells.

CELLS FOUND IN EFFUSIONS Mesothelial Cell Parietal and viscer al surfaces of pleural and peritoneal cavities are lined by a single layer of flat mesothelial cells. They have basophilic cytoplasm, round or oval nuclei, reticulated chromatin, and usually one or more nucleoli (Fig. lF). During periods of transudative fluid accumulation in the cavities m esothelial cells hypertrophy, the cell layer undergoes hyperplasia, and individual cells exfoliate into the cavity fluid. Mesoth elial cells are large and are frequently encountered in larger number on the feathered edge of fluid smears. They tend to disap pear or to be absent in exudative fluids. Reactive Mesothelial Cells. Reactive m esothelial cells h ave dee p blue cytoplasm, round or oval nuclei with stippled chromatin , and u sually one or m ore nucleoli (Fig. 1, A ,B,E,G) . They have fine p seudopodia or hair-like cytoplasmic processes difficult to discern by light microscopy; occasionally these processes are accentuated by drying smears too

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slowly and appear as an eosinophilic fluffy halo surrounding the cell (Fig. 1, A,F,G) .11 A thin perinuclear zone of pallor (Fig. lA ) and cytoplasmic granules (Fig. lB) b elieved to be glycogen 11 may be observed. Binucleated or multinucleated reactive m esothelial cells and mitotic figures (Fig. 1, B,E,G,) may be found as well as small clumps or aggregates of cells (Fig. lE). Given these findings , confusion with neoplastic cells is a common problem. Mesothelial cell clumps are usually associated with a continuous series of similar aggregates of diminishing size down to pairs and singles, whereas malignant cell clumps are usually not connected to individual cells by inter mediate sized aggregates. 11 Differentiation of reactive mesothelial cells and malignant cells by the periodic acid-Schiff (PAS) staining reaction is not often possible because certain malignant epithelial cells may also produce PAS-positive material. Transformed Mesothelial Cells. Transformed mesothelial cells are derived from reactive forms and cannot be distinguished from macrophages. Intermediate forms bearing some characteristics of reactive forms may be identified. These mesothelial cells have a p ale blue cytoplasm which is often granulated and occasionally vacuolated (Fig. 1,

Figure 2. Modified tra nsudates or hemorr hage. (Wright's stain, X 1200.) A, Low magnification of neoplastic cell aggregate ; nondegenerat.e ·neutrophils and red blood cells may be seen ( x 300) . B, Adenocarcinoma cell aggregate illustr a ting anisokaryosis. C, Adenocarcinoma cell aggregat e illus tra ting nuclear molding and giant nucleolus. D , "Jeoplastic lymphocytes , a nondegenerate neutrophil, and red blood cells (feline lymph o sarcoma). E, Small lymphocytes and nondegenerate neutrophils with a smudged cell and several lipid droplets (felin e chylothorax). F, Active hemorrhagic e ffusion with red blood cells, le ukocytes, a nd a large platelet. aggregate ( lower center). G, C hronic or r esol ving hemorr h agic effus ion with rnacr ophages containing red blood cells and h e mosiderin , non d egenerate neutrophils, and reel blood cells.

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Figure 3. Exudates (Wright"s stain, x 1200.) A , Non septic exudate- bile-induced peritonitis with three reactive mesthelial cells (upper right), a macrophage filled with biliary pigment (center left), and nondegenerate neutr ophils. B, Nonseptic exudate with nondegenerate neutrophi ls and r ed blood cells. C, '\onseptic exudate with n ondegenerate ne u trop hils and macro phages. D , Macrophages containing phagocytosed cell deb ris, nondegenerate neutrophils, and a uniforml y dispersed proteinacious precipitate in the background (feline infectious per itonitis). E, Two nondegener ate neutrophils (one very p yk notic a nd rounded) and beaded bra nchin g fila mentou s bacteria comp atibl e with Actin om yces sp . F, Septic exudate with numerous karvolvtic (d egenerate) n e utrophils, · o~ca­ sional p yknotic and karyorrhectic ne u trophils, and macrophages. G, Se pt ic exudate fro m inte st ina l perforation-pe ritoniti s with degenerate neu trophils, one small lym p h ocyte (upper right) and a variety of bacteria.

D,E). Their nuclear chromatin is more clumped than that of reactive

forms. They may be phagocytic occasionally containing erythrocytes, other cells, or cell debris in their cytoplasm which makes differentiation from monocyte-derived m acrophages arbitrary and m eaningless. Occasionally transformed forms become greatly swollen by giant vacuolization which pushes their nuclei to the cell edge (Fig. IC).

Macrophage Macrophages are derived from blood monocytes. They have pale blue granulated cytoplasm which is often vacuolated and in cer tain circumstances contains phagocytized cells, cell d ebris, bacteria, or pigments (Figs. 2G and 3, A,C,D). Macrophages occur in low number in chronic exudative effu sions and may be present in certain tran sudative fluids and chronic hemorrhage (Fig. 2G) as well.

Neutrophil Neutrophils are the p redominant nucleated cell type in many transudative and most exud ative effusions. They are typically nondegenerate

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in transudates, nonseptic exudates, and certain septic exudates in which the etiologic agent is of low toxicity or is a nontoxin producer. Neutrophils of most septic exudates are degenerate. Time delay in making smears causes most neutrophils to become degenerate. Nondegenerate Neutrophils. Nondegenerate neutrophils appear to be similar to those of blood, but may have more nuclear lobes and pyknosis with densely aggregated chromatin (Fig. 3, A-E). Occasionally the nuclei appear to be rounded which makes the cell difficult to differentiate from a lymphocyte (Fig. 3E). The cytoplasm may appear as that of blood neutrophils or be more basophilic. If nondegenerate neutrophils predominate in an effusion, consideration should first be given to transudative or nonseptic exudative causes. Occasionally, however, bacteria may be observed or cultured; Actinomyces species-induced exudative effusions are characterized in the early stages by predominantly nondegenerate neutrophils. Degenerate Neutrophils. In most septic exudates neutrophil nuclei undergo karyolysis characterized by swelling, pale pink staining chromatin, and dissolution of the nucleus (Fig. 3, F,G). Observation of karyolytic neutrophils is a major signalment to search for bacteria by observation or culture. The cytoplasm of degenerate neutrophils may show toxic granulation or vacuolization. Another form of degenerate neutrophil is that which has undergone karyorrhexis in which the pyknotic nucleus has broken up into several dark round pieces (Fig. 3F). A few karyorrhectic neutrophils can be found in any effusion in which either degenerate (karyolytic) or nondegenerate neutrophils predominate. Lymphocyte Lymphocytes predominate in early stages of chylothorax, pseudochylous thoracic effusion of cardiac disease in cats, and may be found in low number in any modified transudate or exudative effusion. They are small round cells with a scanty rim of cytoplasm visible around the nucleus; the nuclear chromatin is smooth or finely stippled (Figs. lB, 2E, and 3G). Nucleoli may be visible using new methylene blue stain but tend to remain obscure in Romanowsky stained preparations. A certain percentage of these cells are lysed during preparation of smears; smudges of purple chromatin are observed (Fig. 2E). Neoplastic Cells The neoplasm most frequently diagnosed by cytologic examination of effusions is lymphosarcoma. Metastatic carcinomas are an infrequent cause of thoracic or abdominal effusion and exfoliated cells may or may not be found on cytology. Sarcomas other than lymphosarcoma may likewise induce a body cavity effusion but individual or aggregates of cells rarely exfoliate.

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Table 1. Cytomorphologic Criteria for Malignancy General Criteria Large cell size-often the first feature observed Aggregates of cells Monomorphism-belong to the same cell line Pleomorphism-variability in cell size and shape Nuclear Criteria High nuclear-cytoplasmic ratio--large nuclei Anisokaryosis-variability in nuclear size Multinucleation of individual cells Normal or abnormal mitotic figures Nuclear molding or deformation by adjacent cells Nucleoli enlarged or variable in size or shape Thickened nuclear membrane-(requires Papanicolaou stain) Nuclear chromacenters vary in size or shape-(requires Papanicolaou stain) Cytoplasmic Criteria ' H yperchromasia Cytoplasmic inclusions-Phagocytosed materials (cells, cell debris) or secretory products (mucin, melanin)

Neoplastic Lymphocytes. Neoplastic lymphocytes of dogs and cats are most often prolymphocytes or lymphoblasts. These variably sized cells are large and round with intensely basophilic cytoplasm (Fig. 2D). Nucleoli are prominent and may vary in number among cells. Carcinoma or Sarcoma Cells. The primary difficulty in identification of neoplastic cells (Fig. 2, A-C) other than those of lymphosarcoma is differentiation from reactive mesothelial cells, particularly aggregates (see "Mesothelial Cell"). Several authors 5 •10 •11 have given cytomorphologic criteria of malignancy which are listed in Table 1. Several of these criteria should be satisfied before an opinion of neoplasia is expressed because any criterion, alone, may be observed among normal cells. If suspicion of neoplasia is rendered by cytologic examination further diagnostic procedures are justified. Neoplasia is a common cause of body cavity effusion but in the majority of instances neoplastic cells are not observed in routine examination of the effusate. Other Cells Other cells encountered in effusions include erythrocytes (red blood cells), eosinophils, mast cells, and plasma cells. Occasionally, during stages of active hemorrhage, platelets may also be observed. Erythrocytes may appear to be similar to those of blood, but are often misshapen and stain bluish or greenish (Fig. 1, A,B,C,E). They are frequently found phagocytized by macrophages (Fig. 2G). Their mere presence does not imply hemorrhage as the vast majority of body cavity effusions, regardless of cause or type, are pink or red on gross inspection and contain variable numbers of red blood cells. Effusion red blood cell counts have been found to be nonspecific and of no diagnostic usefulness in man, 2 and the same appears to be true in animals.

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Table 2. Body Cavity Effusion Data from 87 Canine and Feline Cases* REFRACTOMETRY

Specific Gravity Mean Range

EFFUSION DIAGNOSIS AND FINAL DIAGNOSIS

Species, Disease (No. Cases)

> 0 z

NUCLEATED CELL COUNT

Protein Mean Range

0

;!.;

Mean

Range

COMMENT

"'0 '"'1

Transudate

C. Hypoproteinemiat

(14)

1.007

1.005-1.016

N.D.



t""

M

::J c

"'0

z "'

3,800-110,000 Bacteria observed or cultured in all I 0 cases. N.D. Bacteria observed or cultured in both cases. N.D.

Both cases in shock from trauma.

*C = canine; F = feline; N.D. = not determined; < = less than; - = approximately. tProtein-losing enteropathy (10); glomerulonephritis (2); chronic liver disease (2). fAbdominal neoplasia (4); pulmonary neoplasia(!); diaphragmatic hernia (2); immune-mediated (I); ruptured urinary bladder, acute (I); bile duct rupture, acute (I). §Surgical-sponge induced peritonitis(!); bile duct rupture, chronic (3); pulmonary neoplasia (I); lung torsion (I); chronic chylothorax (3).

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Eosinophils are infrequently encountered, especially in transudative effusions, and usually accompany concomitant circulating eosinophilia. The occasionally observed mast cell and plasma cell have little diagnostic significance.

DIFFERENTIAL DIAGNOSIS OF EFFUSIONS Transudates

Transudates are caused primarily by osmotic pressure alterations resulting from hypoproteinemia (hypoalbuminemia) which occurs in chronic renal, hepatic, or enteric diseases. The effusion is most frequent in the abdomen but may be found in the pleural cavity as well. It is usually colorless and clear. Results of refractometry (specific gravity and protein estimate) and nucleated cell count on 14 cases are given in Table 2. Nondegenerate neutrophils and reactive and transformed mesothelial cells are the major cell types in the sparsely cellular fluid. Modified Transudates

Canine Cardiac Disease. Cardiac insufficiency in dogs is a common cause of ascites. The fluid is usually pink to red and cloudy. Refractometry and nucleated cell count data from 9 cases is summarized in Table 2. The predominant nucleated cell type is the nondegenerate neutrophil. Reactive and transformed mesothelial cells and variable numbers of red blood cells are constant components. Lymphocytes, macrophages, and eosinophils (especially with dirofilariasis) may also be observed. Miscellaneous Causes. A variety of disorders in the dog may cause abdominal or pleural effusions which mimic those of cardiac disease. These include pulmonary or abdominal neoplasia, diaphragmatic hernia, and early stages of urinary bladder and bile duct or gallbladder rupture. These fluids are likewise pink or red but may be yellow (bile or urine) and are usually cloudy. Data from 10 cases are shown in Table 2; cell counts are frequently slightly higher than expected with cardiac insufficiency disease. Cytology is very similar to that of cardiac disease and further analyses and diagnostic procedures are necessary for definitive diagnosis. Modified transudates of neoplasia-induced effusions only rarely contain exfoliated neoplastic cells. The effusions of bile duct (Fig. 3A) or gallbladder rupture and of urinary bladder rupture tend to become more cellular (nondegenerate neutrophils and macrophages) with time as subsequent inflammation ensues. Golden-brown bile precipitate or phagocytized bilirubin, a greenish-brown or black pigment, may be observed. Urinary crystals may likewise be clues to diagnosis. Bilirubin or urea nitrogen assay is seldom useful because rapid effusion-plasma equilibration occurs following rupture.

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Chylothorax. Ruptured thoracic duct and chylothorax occurs in dogs and cats. The gross appearance of the effusion is opaque or milky, pink, or white, and is easily confused as a purulent exudate. Nucleated cell counts and refractometry on the fluid supernate yield values (Table 2) characteristic of modified transudate. The supernate usually remains opaque (lipid) and the refractometry values are erroneously high. The presence of lipid in the supernate may be confirmed by the following procedure. 1 An aliquot of supernate is added to each of two tubes which contain 1 or 2 drops of 1 N sodium hydroxide; an equal volume of ether is added to one tube and an equal volume of water is added to the second tube; after mixing the ether-containing tube will be clear if the effusate contains lipid. Smears stained with new methylene blue may reveal lipid droplets or chylomicra as small refractile bodies between cells or adherent to cell surfaces. These droplets are more difficult to observe on Romanowsky stained smears (Fig. 2E). Variable numbers of nondegenerate neutrophils as well as low numbers of mesothelial cells, macrophages, and red blood cells also occur. With time the cellular pattern begins to change with increasing numbers of nondegenerate neutrophils and macrophages. This results from inflammation and the lymphopenia produced by repeated drainage of the cavity. Feline Cardiac Disease-Pseudochylous Effusion. Cardiac disease is frequently a cause of pleural effusion in cats. Pure transudate and modified transudate (cytologically similar to the abdominal effusion of canine cardiac disease) may be observed. However, the pleural effusion of feline cardiac disease is frequently indistinguishable from chylothorax. 1 In our laboratory eight cases were originally diagnosed as chylothorax based on cytology, presence oflipid droplets or chylomicra, cell counts, and refractometry (see Table 2 under pseudochylous-cardiac disease). Each cat was subsequently found to have cardiac disease (cardiomyopathy, endocarditis with saddle thrombus, dirofilariasis 3 ), and a disrupted thoracic duct was not found at necropsy in any case. Lymphosarcoma. Thymic or mediastinal and occasionally abdominal lymphosarcoma may cause effusion in dogs and cats. The fluids may be white, pink, or red and are usually cloudy or opaque. Nucleated cell counts and refractometry values (Table 2) are similar to other types of modified transudates although slightly higher. The diagnosis is readily made upon finding neoplastic lymphocytes (described previously) in stained smears (Fig. 2D). Many lysed cells and variable numbers of nondegenerate neutrophils, mesothelial cells, macrophages, and red blood cells may be present. Occasionally simultaneous chylothorax may be identified in lymphosarcoma-pleural effusions. Nonseptic Exudates Exudative effusions are somewhat arbitrarily divided into nonseptic and septic exudate on the basis of the presence of nondegenerate neu-

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trophils or the absence of bacteria in the former and the presence of degenerate neutrophils or bacteria in the latter. Both types are characterized by very high nucleated cell counts and by refractometry values similar to those of modified transudates (Table 2). As previously mentioned, nonseptic exudative effusions may develop in chronic stages of chylothorax, bile duct, or gallbladder rupture and urinary bladder rupture which were originally characterized as modified transudates. Other causes may include nonseptic inflammation of the pleura or peritoneum secondary to neoplasia, various intestinal obstructions, diaphragmatic hernia, torsion of various organs or sterile foreign bodies. Nonseptic effusions may become contaminated and be characterized on subsequent examinations as septic exudates. Nonseptic exudates may appear white, pink, or red and are nearly always opaque. Nondegenerate neutrophils predominate; a few pyknotic and karyorrhectic nuclei may be observed however. Variable numbers of macrophages, lymphocytes, and red blood cells are usually present (Fig. 2, A-C). Feline Infectious Peritonitis. Feline infectious peritonitis causes a pleural or peritoneal nonseptic exudative effusion which may be readily differentiated from other body cavity effusions. The effusate is quite viscid, usually yellow with an occasional green irridescence, and clear or slightly cloudy. Nucleated cell counts are lower than other nonseptic exudates and similar to modified transudates (Table 2). The protein concentration is often only slightly less than serum protein concentration. Globulin assay or effusion-protein electrophoresis reveals high globulin content. Nondegenerate neutrophils and macrophages predominate on stained smears. A faintly eosinophilic, granular, proteinaceous precipitate is dispersed uniformly across the smear (Fig. 3D). Other cells present may include low numbers of lymphocytes and rare plasma cells. Red blood cells are in small numbers in effusions in feline infectious peritonitis. Septic Exudates

Septic exudates are differentiated from nonseptic exudates by observing degenerate neutrophils or isolating or observing bacteria. Bacterial species which may be isolated include a variety of gram-positive or gram-negative aerobic or anaerobic organisms. Fungi are rarely encountered. Sepsis may occur by external puncture wounds, septic foreign body, or intestinal perforation or rupture. Frequently effusions originally diagnosed as nonseptic exudates (see causes described above) become contaminated and emerge as septic exudates. Septic exudates may appear white, yellow, yellow-greenish, or redtinged and opaque with flocculent particles. Nucleated cell counts are typically very high but may be erroneous if cellular degeneration is ex-

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cessive. Refractometry values are similar to those of nonseptic exudates and modified transudates (Table 2). Degenerate neutrophils predominate on stained smears and macrophages may be present in variable numbers; occasional lymphocytes may be observed (Fig. 3, F,G). Bacteria, which stain blue-black with Romanowsky stains, may be observed intracellularly or extracellularly. Gut perforation or rupture-induced peritonitis usually contains a mixed pleomorphic bacterial population (Fig. 3G). Actinomyces Species-Induced Exudate. Exudative effusions caused by Actinomyces sp. are classified as septic, but in early stages of the disease may appear nonseptic in character. Neutrophils are typically nondegenerate in pure Actinomyces sp. infections. Furthermore the organisms tend to colonize within the effusion; colonies may be fortuitously and unfortunately missed during sampling or during examination of smears. When observed the organisms are found to be fine, beaded, and branching filaments (Fig. 3£). Quite often observation of these organisms on stained smears is not substantiated by successful isolation and cultural identification.

Hemorrhagic Effusions Hemorrhagic effusions are perhaps the most difficult to specifically diagnose cytologically. Many of the transudative and exudative effusions are also red; any effusion may clot or contain clots. Nucleated cell counts and refractometry values on hemorrhagic effusions do not differentiate but are similar to modified transudates (Table 2). The diagnosis must be based on circumstantial evidence as well as effusion evaluation in many instances. History of trauma, shock, or recent surgical intervention of internal vasculature causes suspicion of hemorrhage. Smears of effusions sampled during active hemorrhage reveal red blood cells and leukocytes with morphology and proportions similar to those of fresh blood (Fig. 2F). The presence of platelets, which are almost never observed in transudates or exudates, may be observed during active hemorrhage. Chronic or resolving hemorrhagic effusions contain no platelets and macrophages with phagocytosed red blood cells and hemosiderin may be numerous (Fig. 2G).

REFERENCES 1. Creighton, S. F., and Wilkins, R. J.: Thoracic effusions in the cat: Etiology and diagnostic features. J.A.A.H.A., 11:66, 1975. 2. Dines, D. E., Pierre, R. V., and Franzen, S. J.: The value of cells in the pleural fluid in the differential diagnosis. Mayo Clin. Proc., 50:571, 1975. 3. Donahoe,]. M., Kneller, S. K., and Thompson, P. E.: Chylothorax subsequent to infection of cats with Dirofilaria immitus. J.A.V.M.A., 164:1107, 1974.

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4. Ettinger, S. J.: Pericardiocentesis. VET. CuN. NoRTH AM., 4:403, 1974. 5. Nieburgs, H. E.: Diagnostic Cell Pathology in Tissues and Smears. New York, Grune and Stratton, 1967. 6. Perman, V.: Transudates and exudates. In Kaneko, J. J., and Cornelius, C. E. (eds.): Clinical Biochemistry of Domestic Animals, Vol II. New York, Academic Press, 1971, Ch. 8. 7. Perman, V., Osborne, C. A., and Stevens, J. B.: Laboratory evaluation of abnormal body fluids. VET. CLIN. NoRTH AM., 4:255, 1974. 8. Schall, W. D.: Thoracentesis. VET. CuN. NoRTH AM., 4:395, 1974. 9. Scott, R. C., Wilkins, R. J., and Greene, R. W.: Abdominal paracentesis and cystocentesis. VET. CLIN. NoRTH AM., 4:413, 1974. 10. Soderstrom, N.: Fine Needle Aspiration Biopsy. New York, Grune and Stratton, 1966. 11. Spriggs, A. 1., and Boddington, M. M.: The Cytology of Effusions. Edition 2. New York, Grune and Stratton, 1968. Department of Veterinary Pathology College of Veterinary Medicine University of Georgia Athens, Georgia 30602